The present invention is directed to DNA polymerases, and more particularly, to a novel formulation of DNA polymerases, which formulation of enzymes is capable of efficiently catalyzing the amplification by PCR (the polymerase chain reaction) of unusually long and faithful products.
DNA polymerase obtained from the hot springs bacterium Thermus aquaticus (Taq DNA polymerase) has been demonstrated to be quite useful in amplification of DNA, in DNA sequencing, and in related DNA primer extension techniques because it is thermostable. Thermostable is defined herein as having the ability to withstand temperatures up to 95xc2x0 C. for many minutes without becoming irreversibly denatured, and the ability to polymerize DNA at high temperatures (60xc2x0 to 75xc2x0 C.). The DNA and amino acid sequences described by Lawyer et al., J. Biol. Chem. 264:6427 (1989), GenBank Accession No. J04639, define the gene encoding Thermus aquaticus DNA polymerase and the enzyme Thermus aquaticus DNA polymerase as those terms are used in this application. The highly similar DNA polymerase (Tfl DNA polymerase) expressed by the closely related bacterium Thermus flavus is defined by the DNA and amino acid sequences described by Akhmetzjanov, A. A., and Vakhitov, V. A. (1992) Nucleic Acids Research 20:5839, GenBank Accession No. X66105. These enzymes are representative of a family of DNA polymerases, also including Thermus thermophilus DNA polymerase, which are thermostable. These enzymes lack a 3xe2x80x2-exonuclease activity such as that which is effective for editing purposes in DNA polymerases such as E. coli DNA polymerase I, and phages T7, T3, and T4 DNA polymerases.
Gelfand et al., U.S. Pat. No. 4,889,818 describe a wild-type (abbreviation used here: WT), native Thermus aquaticus DNA polymerase. Gelfand et al., U.S. Pat. No. 5,079,352 describe a recombinant DNA sequence which encodes a mutein of Thermus aquaticus DNA polymerase from which the N-terminal 289 amino acids of Thermus aquaticus DNA polymerase have been deleted (claim 3 of ""352, commercial name Stoffel Fragment, abbreviation used here: ST), and a recombinant DNA sequence which encodes a mutein of Thermus aquaticus DNA polymerase from which the N-terminal 3 amino acids of Thermus aquaticus DNA polymerase have been deleted (claim 4 of ""352, trade name AmpliTaq, abbreviation used here: AT). Gelfand et al. report their muteins to be xe2x80x9cfully activexe2x80x9d in assays for DNA polymerase, but data as to their maximum thermostability is not presented.
Amplification of DNA spans by the polymerase chain reaction (PCR) has become an important and widespread tool of genetic analysis since the introduction of thermostable Taq DNA polymerase for its catalysis. However, one remaining limitation to prior art methods of PCR is the size of the product span that can be amplified. For full-length Taq DNA Polymerase and for N-terminally truncated variants such as Klentaq-278, Klentaq5 and Stoffel Fragment, PCR amplification apparently rapidly becomes inefficient or non-existent as the length of the target span exceeds 5-6 kb. This was shown even when 30 minutes was used during the extension step of each cycle.
Although there are several reports of inefficient but detectable amplification at 9-10 kb target length and one at 15 kb, most general applications are limited to 5 kb.
Kainze et al. (Analytical Biochem. 202:46-49(1992)) report a PCR amplification of over 10 kb: a 10.9 kb and a 15.6 kb product, utilizing an enzyme of unpublished biological source (commercially available as xe2x80x9cHot Tubxe2x80x9d DNA polymerase). Kainze et al. report achieving a barely visible band at 15.6 kb after 30 cycles, starting with 1 ng of xcex DNA template per 100 ul of reaction volume. The efficiency of this amplification was shown to be relatively low, although a quantitative calculation of the efficiency was not presented. Attempts by Kainze et al. to make WT Thermus aquaticus DNA polymerase perform in the 10-15 kb size range were not successful, nor have successful results been reported by anyone else for any form of Thermus aquaticus DNA polymerase in this size range.
A DNA polymerase formulation capable of efficient amplification of DNA spans in excess of 6 kb would significantly expand the scope of applications of PCR. For instance, whole plasmids, and constructs the size of whole plasmids, could be prepared with this method, which would be especially valuable in cases in which a portion of the DNA in question is toxic or incompatible with plasmid replication when introduced into E. coli. If this thermostable DNA polymerase preparation simultaneously conferred increased fidelity to the PCR amplification, the resulting large products would be much more accurate, active and/or valuable in research and applications, especially in situations involving expression of the amplifed sequence. If the thermostable DNA polymerase preparation allowed, in addition, more highly concentrated yields of pure product, this would enhance the method of PCR to the point where it could be used more effectively to replace plasmid replication as a means to produce desired DNA fragments in quantity.
Among the several objects of the invention, therefore, may be noted the provision of a formulation of DNA polymerases capable of efficiently catalyzing primer extension products of greater length than permitted by conventional formulations, including lengths up to at least 35 kilobases, that reduces the mutagenicity generated by the PCR process, particularly in comparison with prior art DNA polymerases and for any target lengths, that maximizes the yield of PCR target fragments and, concomitantly, enhances the intensity and sharpness of PCR product bands, without significant sacrifice in flexiblity, specificity, and efficiency; and the provision of an improved process for amplification by PCR which can be utilized to reliably synthesize nucleic acid sequences of greater length and which can effectively utilize PCR products as primers.
Briefly, therefore, the present invention is directed to a novel formulation of thermostable DNA polymerases including at least one thermostable DNA polymerase lacking 3xe2x80x2-5xe2x80x2 exonuclease activity and at least one thermostable DNA polymerase exhibiting 3xe2x80x2-5xe2x80x2 exonuclease activity.
In another aspect, a kit for the synthesis of a polynucleotide is provided, comprising a first DNA polymerase which posesses 3xe2x80x2-5xe2x80x2 exonuclease activity, and a second DNA polymerase which lacks 3xe2x80x2-5xe2x80x2 exonuclease activity.
In another aspect, a kit for the synthesis of a polynucleotide is provided, comprising a first DNA polymerase which posesses 3xe2x80x2-5xe2x80x2 exonuclease activity, and a second DNA polymerase which lacks 3xe2x80x2-5xe2x80x2 exonuclease activity, wherein the first DNA polymerase is selected from the group consisting of Pyrococcus furiosus DNA polymerase, Thermotoga maritima DNA polymerase, Thermococcus litoralis DNA polymerase, and Pyrococus GB-D DNA polymerase, and the second DNA polymerase is selected from the group consisting of Thermus aquaticus DNA polymerase, (exoxe2x88x92) Thermococcus literalis DNA polymerase, (exoxe2x88x92)Pyrococcus furiosus DNA polymerase, and (exoxe2x88x92) Pyrococcus GB-D DNA polymerase.
In a further embodiment of the invention, a method of amplifying a polynucleotide sequence is provided. The method includes the steps of mixing a composition with a synthesis primer, and a synthesis template, with the composition including a first DNA polymerase possessing 3xe2x80x2-5xe2x80x2 exonuclease activity, and a second DNA polymerase lacking 3xe2x80x2-5xe2x80x2 exonuclease activity.
In yet another aspect of the invention, a method of amplifying a polynucleotide sequence is provided. The method includes the steps of mixing a composition with a synthesis primer, and a synthesis template, with the composition including a first DNA polymerase possessing3xe2x80x2-5xe2x80x2 exonuclease activity which is selected from the group consisting of Pyrococcus furiosus DNA polymerase, Thermotoga maritima DNA polymerase, Thermococcus litoralis DNA polymerase, and Pyrococcus GB-D DNA polymerase, and a second DNA polymerase lacking 3xe2x80x25 exonuclease activity which is selected from the group consisting of Thermus aquaticus DNA polymerase, (exoxe2x88x92) Thermococcus litoralis DNA polymerase, (exoxe2x88x92) Pyrococcus furiosus DNA polymerase, and (exoxe2x88x92) Pyrococcus GB-D DNA polymerase.
Other objects and features will be in part apparent and in part pointed out hereinafter.
The listed abbreviations and terms, as used herein, are defined as follows:
Abbreviations:
bp=base pairs
kb=kilobase; 1000 base pairs
nt=nucleotides
BME=beta-mercaptoethanol
PPi=sodium pyrophosphate
In use, the following 3-letter abbreviations often refer to the single-chain DNA polymerase elaborated by the microorganism.
Pfu=Pyrococcus furiosus 
Pwo=Pyrococcus woesii 
Taq=Thermus aquaticus 
Tfl=Thermus flavus 
Tli=Thermococcus literalis 
Klentaq-nnn=N-terminally deleted Thermus aquaticus DNA polymerase that starts with codon nnn+1, although that start codon and the next codon may not match the WT sequence because of alterations to the DNA sequence to produce a convenient restriction site.
WT=wild-type (full length) or deletion of only 3 aa
aa=amino acid(s)
ST=Stoffel fragment, an N-terminal deletion of Thermus aquaticus DNA polymerase that could be named Klentaq-288.
LA=Long and Accurate; an unbalanced mixture of two DNA polymerases, at least one lacking significant 3xe2x80x2-exonuclease activity and at least one exhibiting significant 3xe2x80x2-exonuclease activity.
PCR=(noun)
1. The Polymerase Chain Reaction
2. One such reaction/amplification experiment.
3.(verb) To amplify via the polymerase chain reaction.
ul=microliter(s)
ATCC=American Type Culture Collection
Megaprimer=double-stranded DNA PCR product used as primer in a subsequent PCR stage of a multi-step procedure.
Deep Vent=DNA polymerase from Pyrococcus species GB-D; purified enzyme is available from New England Biolabs.
Deep Vent exoxe2x88x92=mutant form of Deep Vent DNA polymerase lacking 3xe2x80x2 (editing)-exonuclease.
Vent=DNA polymerase from Thermococcus litoralis; purified enzyme is available from New England Biolabs.
Vent exoxe2x88x92=mutant form of Vent DNA polymerase lacking 3xe2x80x2 (editing)-exonuclease.
Pfu=DNA polymerase from Pyrococcus furiosus lacking 3xe2x80x2 (editing)-exonuclease; purified enzyme is available from Stratagene Cloning Systems, Inc.
Pfu exoxe2x88x92=mutant form of Pfu DNA polymerase purified enzyme is available from Stratagene Cloning Systems, Inc.
SEQUENASE=A chemically modified or a mutated form of phage T7 or T3 DNA polymerase wherein the modification or mutation eliminates the 3xe2x80x2-exonuclease activity.
THESIT=polyethylene glycol monododecyl ether.